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WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

PCT

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 6
A61K 31/79, 31/40, 33/24

(11) International Publication Number: WO 97/40842

(43) International Publication Date: 6 November 1997 (06.1 1.97)

(21) International Application Number:

(22) Internationa] Filing Date:

PCT/US97/07801
May 1997 (01.05.97)

(30) Priority Data:

60/016,658
08/841,738

1 May 1996 (01.05.96) US
30 April 1997 (30.04.97) US

(71) Applicant (for all designated States except US): ELI LILLY

AND COMPANY [US/US]; Lilly Corporate Center, Indi-
anapolis, IN 46285 (US).

(72) Inventors; and

(75) Inventors/Applicants (for US only): JIROUSEK, Michael, R.
[US/US]; 10342 Fawn Ridge Lane, Indianapolis, IN 46236
(US). STRAMM, Lawrence, E. [US/US]; 9143 Hadway
Drive, Indianapolis, IN 46256 (US). WAYS, Douglas, Kirk
[US/US]; 4565 North Park Avenue, Indianapolis, IN 46205
(US).

(74) Agents: SKERPON, Joseph, M. et al.; Banner & Witcoff, Ltd.,
Suite 1100, 1001 G Street, N.W., Washington, DC 20001
(US).

(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, Fl, GB, GE,
GH, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR,
LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ,
PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT,
UA, UG, US, UZ, VN, ARIPO patent (GH, KE, LS, MW,
SD, SZ, UG), Eurasian patent (AM, AZ, BY, KG, KZ, MD,
RU, TJ, TM), European patent (AT, BE, CH, DE, DK, ES,
FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent
(BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD,
TG).

Published

With international search report.

(54) Title: USE OF PROTEIN KINASE C INHIBITORS TO ENHANCE THE CLINICAL EFFICACY OF ONCOLYTIC AGENTS AND
RADIATION THERAPY

(57) Abstract

A method for treating neoplasms is disclosed, particularly using the /3-isozyme selective PKC inhibitor, (S)-3,4-[N,N'-l,l'-((2"-
ethoxy)-3"'(0)-4"'-(N,N-dimethylamino)-butane)-bis-(3,3'-indolyl)]-l(H)-pyrrole-2,5-dioneoroneof its salts, such PKC inhibitors enhance
the clinical efficacy of oncolytic agents and radiation therapy.

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.

Albania

Spain

Lesotho

Slovenia

Armenia

Finland

Lithuania

Slovakia

Austria

France

Luxembourg

Senegal

Australia

Gabon

Latvia

Swaziland

Azerbaijan

United Kingdom

Monaco

Chad

Bosnia and Herzegovina

Georgia

Republic of Moldova

Togo

Barbados

Ghana

Madagascar

Tajikistan

Belgium

Guinea

The former Yugoslav

Turkmenistan

Burkina Faso

Greece

Republic of Macedonia

Turkey

Bulgaria

Hungary

Mali

Trinidad and Tobago

Benin

Ireland

Mongolia

Ukraine

Brazil

Israel

Mauritania

Uganda

Belarus

Iceland

Malawi

United States of America

Canada

Italy

Mexico

Uzbekistan

Central African Republic

Japan

Niger

Viet Nam

Congo

Kenya

Netherlands

Yugoslavia

Switzerland

Kyrgyzstan

Norway

Zimbabwe

C6te d'lvoire

Democratic People's

New Zealand

Cameroon

Republic of Korea

Poland

China

Republic of Korea

Portugal

Cuba

Kazakstan

Romania

Czech Republic

Saint Lucia

Russian Federation

Germany

Liechtenstein

Sudan

Denmark

Sri Lanka

Sweden

Estonia

Liberia

Singapore

97/40842

PCT/US97/07801

USE OF PROTEIN KINASE C INHIBITORS TO ENHANCE THE
CLINICAL EFFICACY OF ONCOLYTIC AGENTS AND RADIATION

THERAPY

This application claims the priority benefits of the U.S. Provisional application
Serial No. 60/016,658 filed May 1, 1996.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is broadly directed to a method for enhancing anti-
neoplasm effects of chemotherapies and radiation therapies with PKC inhibitors.
The present invention is particularly directed to the use of Protein Kinase C (PKC)
inhibitors, especially a particular class of isozyme selective PKC inhibitors in
combination with an oncolytic agent or y-irradiation to enhance their anti-
neoplasm effects in treatment of neoplasms.

2. Description of Related Art

Therapeutic treatments have been developed over the years to treat
neoplasms. There are two major approaches to treat neoplasms: 1) chemotherapy
employing oncolytic agents, and 2) radiation therapy, e.g., Y-irradiation. Oncolytic
agents and v-irradiation cause cytotoxic effects, preferentially to tumor cells, and
cause cell death.

Studies have shown that y-irradiation and certain groups of oncolytic
agents assert their cytotoxic effects by activating programmed cell death or
apoptosis. A balance between the activities of apoptotic and antiapoptotic

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intracellular signal transduction pathways is important towards a cell's decision of
undergoing apoptosis in response to the above mentioned chemotherapy as well as
radiation therapy.

PKC inhibitors has been proposed for cancer therapy, e.g. see U.S. 5,552,391,
and PKC activities have been indicated to exert antiapoptotic effects, especially in
response to radiation therapies, e.g., y-irradiation. In particular, studies have shown that
activation of PKC inhibits apoptosis induced by anti-neoplasm agents such as Ara-c, 2-
chloro-2-deoxyadenosine, 9-P-D-arabinosyl-2-fluoroadenine, and y-irradiation therapy.
There also have been indications that down regulation of PKC activities in tumor cells
enhances apoptosis stimulated by oncolytic agents. PKC activation has been shown to
attenuate y-irradiation induced cell death.

There is a need in the art to develop therapeutic agents which enhance the
apoptotic signal transduction pathways in cells and thereby enhance the clinical efficacy
of oncolytic agents and radiation therapy.
SUMMARY OF INVENTION

It is an object of the invention to provide methods for treating a neoplasm.
It is another object of the invention to provide methods for enhancing an anti-
neoplasm effect of an oncolytic agent.

It is still another object of the invention to provide methods for enhancing anti-
neoplasm effects of radiation therapy.

These and other objects of the invention are provided by one or more of the
embodiments described below.

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In one embodiment of the invention there is provided a method for treating
a neoplasm which comprises administrating to a mammal in need of such treatment
an oncolytic agent or y-irradiation in combination with a protein kinase C
inhibitor.

In still another embodiment of the invention there is provided a method for
enhancing an anti-neoplasm effect of chemotherapy and radiation therapy which
comprises administrating a protein kinase C inhibitor in combination with said
oncolytic agent or radiation therapy.

The present invention provides the art with a method for increasing
apoptotic effects in cells and is thus effective in enhancing the anti-neoplasm
effects of chemotherapies and radiation therapies.
RRTFF DVSrRTPTTON OF THE DRAWINGS

Figure 1 shows the dosage effect of bryostatin 1 on PKC isoforms.

Figure 2 demonstrates the incubation time effect of bryostatin 1 on PKC
isoforms.

Figure 3 demonstrates that down regulation of PKC-p* enhances the efficacy
of Y-irradiation.

Figure 4 shows that increased expression of PKC-p demonstrates resistance
to radiation stimulated cell death.
DETAILED p raraipnoN of thf invention

It is a discovery of the present invention that use of PKC inhibitors,
especially a particular class of protein kinase C inhibitors, reduces or inhibits anti-

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apoptotic effects in a cell. Consequently, such compounds can be used to enhance
the anti-neoplasm effects of chemotherapies and radiation therapies.

The method of this invention may employ any PKC inhibitor known in the
art including non-specific PKC inhibitors and specific PKC inhibitors of different
isoforms. Informations about PKC inhibitors, and methods for their preparation
are readily available in the art. For example, different kinds of PKC inhibitors and
their preparation are described in U.S. Patents 5621 101, 5621098, 5616577,
5578590, 5545636, 5491242, 5488167, 5481003, 5461146, 5270310, 5216014,
5204370, 5 141957, 4990519, and 4937232, all of which are incoporated herein by
reference. Preferably the present invention utilizes those protein kinase C
inhibitors that effectively inhibit the P isozyme. One suitable group of compounds
are generally described in the prior art as bis-indolylmaleimides or macrocyclic
bis-indolylmaleimides. Bis-indolymaleimides well recognized in the prior art
inlcude those compounds described in U.S. Patents 5621098, 5552396, 5545636,
5481003, 5491242, and 5057614, all incorporated by reference herein.
Macrocyclic bis-mdolytaialeimides are particularly represented by the compounds
of formula I. These compounds, and methods for their preparation, have been
disclosed in U.S. Patent 5,552,396, which is incorporated herein by reference. In
accordance with the present invention, these compounds are administered in
combination with other anti-neoplasm therapies to a mammal in need of such
treatment. In particular, these compounds can be used to enhance the anti-
neoplasm effects of chemotherapies and radiation therapies.

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One preferred class of compounds for use in the method of the invention
has the formula:

R 2

(I)

wherein:

W is -O-, -S-, -SO-, -SO r , -CO-, C 2 -C 6 alkylene, substituted alkylene, C 2 -
C 6 alkenylene, -aryl-, -aryl(CH 2 ) m O-, -heterocycle-, -heterocycle-(CH 2 ) m O-, -fused
bicyclic-, -fused bicyclic-(CH 2 ) m O-, -NR 3 -, -NOR 3 -, -CONH-, or -NHCO-;

X and Y are independently C r C 4 alkylene, substituted alkylene, or together
X, Y, and W combine to form -(CH 2 )„-AA-;

R's are hydrogen or up to four optional substituents independently selected
from halo, C,-C 4 alkyl, hydroxy, C ,-C 4 alkoxy, haloalkyl, nitro, NR 4 R 5 , or -

NHCO(C,-C 4 alkyl);

R 2 is hydrogen, CH 3 CO-, NH 2 , or hydroxy;

R 3 is hydrogen, (CH 2 ) m aryl, C,-C 4 alkyl, -COO(C r C 4 alkyl), -CONR 4 R 5 , -
(C=NH)NH 2 , -SO(C,-C 4 alkyl), -SO, (NR 4 R 5 ), or -S0 2 (C,-C 4 alkyl);

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R 4 and R 5 are independently hydrogen, C,-C 4 alkyl, phenyl, benzyl, or
combine to the nitrogen to which they are bonded to form a saturated or
unsaturated 5 or 6 member ring;

AA is an amino acid residue;

m is independently 0, 1, 2, or 3; and

n is independently 2, 3, 4, or 5, or a pharmaceutically acceptable salt,

prodrug or ester thereof.

A more preferred class of compounds for use in this invention is
represented by formula I wherein the moieties -X-W-Y- contain 4 to 8 atoms,
which may be substituted or unsubstituted. Most preferably, the moieties -X-W-Y-
contain 6 atoms.

Other preferred compounds for use in the method of this invention are those
compounds of formula I wherein R l and R 2 are hydrogen; and W is a substituted
alkylene, -O-, S-, -CONH-, -NHCO- or -NR 3 -. Particularly preferred compounds
are compounds of the formula la:

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wherein Z is -(CU 2 %- or -(CH 2 ) p -0-(CH 2 ) p -; R 4 is hydroxy, -SH, C , -C 4 alkyl,
(CH 2 ) m aryl, -NH(aryl), -N(CH 3 ) (CF 3 ), -NH(CF 3 ), or -NR 5 R 6 ; R 5 is hydrogen or C r
C 4 alky; R 6 is hydrogen, C ,-C 4 alkyl or benzyl; p is 0, 1, or 2; and m is
independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester
thereof. Most preferred compounds of the formula la are those wherein Z is CH 2 ;
and R 4 is -NH 2 , -NH(CF 3 ), or -N(CH 3 ) 2 .

Other preferred compounds for use in the method of the present invention
are compounds wherein W in formula I is -O-, Y is a substituted alkylene, and X is
an alkylene. These preferred compounds are represented by formula lb:

wherein Z is -(CH 2 ) P -; R 4 is -NR S R 6 , -NH(CF 3 ), or -N(CH 3 ) (CF 3 ); R 5 and R 6 are
independently H or C,-C 4 alkyl; p is 0, 1, or 2; and m is independently 2 or 3, or a
pharmaceutically acceptable salt, prodrug or ester thereof. Most preferred
compounds of formula lb are those wherein p is 1; and R 5 and R 6 are methyl.

Because they contain a basic moiety, the compounds of formulae I, la, and
lb can also exist as pharmaceutically acceptable acid addition salts. Acids

WO 97/40842 PCT/US97/07801

commonly employed to form such salts include inorganic acids such as
hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acid, as well as
organic acids such as para-toluenesulfonic, methanesulfonic, oxalic, para-
bromophenylsulfonic, carbonic, succinic, citric, benzoic, acetic acid, and related
5 inorganic and organic acids. Such pharmaceutically acceptable salts thus include

sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, mono-
hydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate,
chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate,
formate, isobutyrate, heptanoate, propiolate, oxalate, malonate, succinate, suberate,
10 sebacate, fumarate, maleate, 2-butyne-l,4-dioate, 3-hexyne-2, 5-dioate, benzoate,

chlorobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate,
phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hippurate, p-
hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate,
naphthalene- 1 -sulfonate, naphthalene-2-sulfonate, mandelate and the like.
15 Particularly the hydrochloric and mesylate salts are used.

In addition to phannaceutically-acceptable salts, other salts also can exist.
They may serve as intermediates in the purification of the compounds, in the
preparation of other salts, or in the identification and characterization of the
compounds or intermediates.
20 The pharmaceutically acceptable salts of compounds of formulae I, la, and

lb can also exist as various solvates, such as with water, methanol, ethanol,
dimethylformamide, ethyl acetate and the like. Mixtures of such solvates can also

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be prepared. The source of such solvate can be from the solvent of crystallization,
inherent in the solvent of preparation or crystallization, or adventitious to such
solvent.

It is recognized that various stereoisomeric forms of the compounds of
5 formulae I, la, and lb may exist; for example, W may contain a chiral carbon atom

in the substituted alkylene moiety. The compounds are normally prepared as
racemates and can conveniently be used as such. Alternatively, both individual
enantiomers can be isolated or synthesized by conventional techniques if so
desired. Such racemates and individual enantiomers and mixtures thereof form
10 part of the compounds used in the methods of the present invention.

The compounds utilized in this invention also encompass the
pharmaceutically acceptable prodrugs of the compounds of formulae I, la, and lb.
A prodrug is a drug which has been chemically modified and may be biologically
inactive at its site of action, but which may be degraded or modified by one or
15 more enzymatic or other in vivo processes to the parent bioactive form. This

prodrug likely may have a different pharmacokinetic profile than the parent,
enabling easier absorption across the mucosal epithelium, better salt formation or
solubility, and/or improved systemic stability (an increase in plasma half-life, for
example). Typically, such chemical modifications include the following:
20 1 ) ester or amide derivatives which may be cleaved by

esterases or lipases;

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2) peptides which may be recognized by specific or nonspecific

proteases; or

3) derivatives that accumulate at a site of action through
membrane selection of a prodrug form or a modified prodrug form; or any
combination of 1 to 3, supra . Conventional procedures for the selection and
preparation of suitable prodrug derivatives are described, for example, in H.
Bundgaard, Design of Prodrugs. (1985).

The synthesis of various bis-indole-N-maleimide derivatives is described in
Davis et al. U.S. Patent 5,057,614 and the synthesis of the preferred compounds
suitable for use in this invention are described in the previously identified U.S.
Patent 5,552,396 and in Faul et al. EP publication 0 657 41 1 Al , all of which are
incorporated herein by reference.

One particularly preferred protein kinase C inhibitor for use in the method
of this invention is the compound described in Example 5g ((S)-3,4-[N, N'-l,l'-
((2"-ethoxy)-3 , "(0)-4"'-(N,N-dimethy lamino)-butane)-bis-(3 ,3 '-indoly 1 )]- 1 (H)-
pyrrole-2,5-dione Hydrochloride Salt) of the aforementioned U.S. Patent
5,552,396. This compound is a potent protein kinase C inhibitor. It is selective to
protein kinase C over other kinases and is highly isozyme-selective, i.e., it is
selective for the beta-1 and beta -2 isozymes. Other salts of this compound also
would be favored, especially the mesylate salts.

A preferred mesylate salt can be prepared by reacting a compound of the

formula II

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with methanesulfonic acid in a non-reactive organic solvent, preferably an
organic/water mixture, and most preferably water-acetone. Other solvents such as
methanol, acetone, ethylacetate and mixtures thereof are operable. The ratio of
solvent to water is not critical and generally determined by the solubility of the
reagents. Preferred solvent to water ratios are generally from 0. 1 : 1 to 1 00: 1
solvent to water by volume. Preferably, the ratio is 1 : 1 to 20: 1 and most preferably
5: 1 to 1 0: 1 . The optimal ratio is dependent on the solvent selected and is
preferably acetone at a 9:1 solvent to water ratio.

The reaction usually involves approximately equimolar amounts of the two
reagents, although other ratios, especially those wherein the methanesulfonic acid
is in excess, are operative. The rate of addition of methanesulfonic acid is not
critical to the reaction and may be added rapidly (<5 minutes) or slowly over 6 or
more hours. The reaction is carried out at temperatures ranging from 0°C to reflux.
The reaction mixture is stirred until formation of the salt is complete, as
determined by x-ray powder diffraction and can take from 5 minutes to 12 hours.

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The salts of the present invention are preferably and readily prepared as a
crystalline form. The trihydrate form of the salt may be readily converted to the
monohydrate upon drying or exposure to 20-60% relative humidity. The salt is
substantially crystalline demonstrating a defined melting point, birefringence, and
5 an x-ray diffraction pattern. Generally, the crystals have less than 1 0% amorphous

solid and preferably less than 5% and most preferably less than 1% amorphous
solid.

The mesylate salt is isolated by filtration or other separation techniques
appreciated in the art directly from the reaction mixture in yields ranging from 5'' *

10 to 100%. Recrystallization and other purification techniques known in the art may

be used to further purify the salt if desired.

The PKC inhibitors, including the compounds described above, are used in
combination with conventional anti-neoplasm therapies to treat mammals,
especially humans with neoplasia. The procedures for conventional anti-neoplasm

15 therapies, including chemotherapies, e.g. using oncolytic agents and radiation

therapies e.g., Y-inadiation are known, readily available, and routinely practiced in
the art, e.g., see Harrison's PRINCIPLES OF INTERNAL MEDICINE 1 1th
edition, McGraw-Hill Book Company.

Neoplasia is characterized by abnormal growth of cells which often results

20 in the invasion of normal tissues, e.g., primary tumors or the spread to distant

organs, e.g., metastasis. The treatment of any neoplasia by conventional anti-
neoplasm therapies can be enhanced by the present invention. Such neoplastic

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growth includes but not limited to primary tumors, primary tumors that are
incompletely removed by surgical techniques, primary tumors which have been
adequately treated but which are at high risk to develop a metastatic disease
subsequently, and an established metastatic disease.
5 Specifically, the PKC inhibitors described above can enhance the anti-

neoplasm effects of an oncolytic agent. The wide variety of available oncolytic
agents are contemplated for combination therapy in accordance with present
invention. In a preferred embodiment, oncolytic agents that assert their cytotoxic
effects by activating programmed cell death or apoptosis are used in combination
10 with the described PKC inhibitors. These include but not limited to 1 -0-D-

arabinofuranosylcytosine or Ara-c, etoposide or VP- 16, cis-
diamminedichloroplatinum (II) or c/j-platinum, doxorubicin or adriamycin, 2-
chloro-2-deoxyadenosine, 9-p-D-arabinosyl-2-fluoroadenine, and glucocorticoids.
All the neoplastic conditions treatable with such oncolytic agents can be
15 treated in accordance with the present invention by using a combination of a PKC

inhibitor with one or more oncolytic agents. The oncolytic agents assert the
cytotoxicity or anti-neoplasm effects in a variety of specific neoplastic conditions.
For example, Ara-c is normally used for treatment of childhood-null acute
lymphoid leukemia (ALL), thymic ALL, B-cell ALL, acute myeloid leukemia,
20 acute granulocytic leukemia and its variants, non-Hodgkins lymphoma,

myelomonocytoid leukemia, acute megakaryocytoid leukemia and Burkitt's
lymphoma, Adult-B-ALL, acute myeloid leukemia, chronic lymphoid leukemia,

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chronic myeloid leukemia, and T cell leukemia. VP- 16 is normally used for
treatment of testicular carcinoma, small and large non-small cell lung carcinoma,
Hodgkin's lymphoma, non-Hodgkin's lymphoma, choriocarcinoma, Ewing's
sarcoma, and acute granulocytic leukemia. Cw-platinum can be employed for
treatment of testicular carcinoma, germ cell tumors, ovarian carcinomas, prostate
cancer, lung cancer, sarcomas, cervical cancer, endomermetrial cancer, gastric
cancer, breast cancer, and cancer of the head and neck. 2-Chloro-2-
deoxyadenosine and 9-P-D-arabinosyl-2-fluoroadenine can be used to treat chronic
lymphoid leukemia, lymphomas and hairy cell leukemia. Doxorubicin can be used
to treat acute granulocytic leukemia and its variants, ALL, breast cancer, bladder
cancer, ovarian cancer, thyroid cancer, lung cancer, Hodgkin's lymphoma, non-
Hodgkin's lymphoma, sarcomas, gastric carcinoma, prostate cancer, endometrial
cancer, Wilm's tumor and neuroblastoma. Clinical effects of oncolytic agents in
all neoplastic conditions treatable with oncolytic agents including the ones
discussed above can be potentiated by use of a combination therapy with the
identified PKC inhibitors in accordance with the present invention.

The PKC inhibitors identified in the present invention can also enhance the
anti-neoplasm effects of a radiation therapy. Usually Y-irradiation is used to treat
the site of a solid tumor directly.

Experimental results provided in the present invention demonstrate that the
complete down regulation or loss of protein kinase C-0 is associated with the
synergistical enhancement of the oncolytic induced apoptosis in human leukemic

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cells (Figure 1). Similarly, significant down regulation of protein kinase C-{1 in
U937 human leukemic cells enhances radiation stimulated cell death (Figure 2).
U937 human leukemic cells that overexpress protein kinase C-P demonstrate
resistance to radiation stimulated cell death (Figure 3). These data provide a strong
indication that the PKC inhibitors, especially p isozyme selective inhibitors,
preferably used in accordance with the present invention can enhance tumor killing
or the anti-neoplasm effects of chemotherapies and radiation therapies and improve
clinical responses to these currently used therapeutic modalities.

The PKC inhibitors of the present invention are administered in
combination with other anti-neoplasm therapies including oncolytic agents and
radiation therapy. The phrase "in combination with other therapies" means that the
compounds can be administered shortly before, shortly after, or concurrent with
such other anti-neoplasm therapies. The compounds can be administered in
combination with more than one anti-neoplasm therapy. In a preferred
embodiment, the compounds are a<irwnistered from 2 weeks to 1 day before any
chemotherapy, or 2 weeks to 1 day before any radiation therapy. Alternatively, the
PKC inhibitors can be administered during chemotherapies and radiation therapies.
If administered following chemotherapy or radiation therapy, the PKC inhibitors
should be given within 1 to 14 days following the primary treatments.

One skilled in the art will recognize that the amount of PKC inhibitor to be
administered in accordance with the present invention in combination with other
anti-neoplasm agents or therapies is that amount sufficient to enhance the anti-

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neoplasm effects of oncolytic agents or radiation therapies or that amount sufficient
to induce apoptosis or cell death. Such amount may vary inter alia, depending
upon the size and the type of neoplasia, the concentration of the compound in the
therapeutic formulation, the specific anti-neoplasm agents used, the timing of the
administration of the PKC inhibitors relative to the other therapies, and the age,
size and condition of the patient.

Both in vivo and in vitro tests can be used to assess the amount of the
compounds needed for inducing apoptosis. For example, human leukemic cells
could be exposed in vitro to various concentrations of oncolytic agents, e.g., Ara-c,
or to radiation in the presence or absence of the PKC inhibitor compounds used in
the present invention. Appropriate neoplastic cell types can be chosen for different
oncolytic agents. Other protein kinase C selective inhibitors can also be used for
comparison. At various time points, cells would be examined for viability by
conventional methods or by any means available in the art. Apoptosis or cell death
can be measured by any means known in the art. Cell death can be determined and
quantified via trypan blue exclusion, and reduced clonogenecity in soft agar. As
well understood by those skilled in the technology, apoptosis is a specific mode of
cell death recognized by a characteristic pattern of morphological, biochemical,
and molecular changes including but not limited to, endonucleolysis (DNA ladder),
abnormal DNA breaks, and condensation of chromatin and cytoplasm (condensed
and punctate nuclei). These changes can be readily detected by any means known
in the art, e.g., microscopy; flow cytometric methods based on increased sensitivity

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of DNA to denaturation and altered light scattering properties; DNA fragmentation
as assessed by agarose gel electrophoresis; terminal DNA transferase assay, (TdT
assay), and nick translation assay (NT assay).

In vivo studies can be done using tumor xenografts inoculated into

5 immunocompromised or sygenic animals. After inoculation and growth of the

primary implant, the animals would be treated with the compounds in the present
invention prior to exposure to the desired oncolytic or radiation treatment. The
size of the tumor implant before and after each treatment in the presence and
absence of the compounds in the present invention can be used as an indication of

10 the therapeutic efficacy of the treatment.

Generally, an amount of protein kinase C inhibitor to be administered in
combination with other anti-neoplasm therapies is decided on a case by case basis
by the attending physician. As a guideline, the extent of the neoplasia, the body
weight, and the age of the patient will be considered, among other factors, when

15 setting an appropriate dose. Normally, the PKC inhibitors of the present invention

are expected to potentiate the anti-neoplasm effects of oncolytic agents and
radiation therapy from about 2 fold to about 10 fold.

Generally, a suitable dose is one that results in a concentration of the
protein kinase C inhibitor at the site of tumor cells in the range of 0.5 nM to 200

20 uM, and more usually from 20 nM to 80 nM. It is expected that serum

concentrations of 40 nM to 150 nM should be sufficient in most circumstances.

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To obtain these treatment concentrations, a patient in need of treatment
likely will be administered between about 0.1 mg per day per kg of body weight
and 1 .5 mg per day per kg. Usually, not more than about 1 .0 mg per day per kg of
body weight of protein kinase C inhibitor should be needed. As noted above, the
above amounts may vary on a case-by-case basis.

The compounds of formula I and the preferred compounds of formula la
and lb are preferably formulated prior to administration. Suitable pharmaceutical
formulations are prepared by known procedures using well known and readily
available ingredients. In making the compositions suitable for use in the method of
the present invention, the active ingredient will usually be mixed with a carrier, or
diluted by a carrier, or enclosed within a carrier which may be in the form of a
capsule, sachet, paper or other container. When the carrier serves as a diluent, it
may be a solid, semisolid or liquid material which acts as a vehicle, excipient or
medium for the active ingredient. Thus, the compositions can be in the form of
tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,
solutions, syrups, aerosol (as a solid or in a liquid medium), soft and hard gelatin
capsules, suppositories, sterile injectable solutions and sterile packaged powders
for either oral or topical application.

Some examples of suitable carriers, excipient, and diluents include lactose,
dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphates,
alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose,
polyvinylpyrrolidone, cellulose, water syrup, methyl cellulose, methyl and

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propylhydroxybenzoates, talc, magnesium stearate and mineral oil. The
formulations can additionally include lubricating agents, wetting agents,
emulsifying and suspending agents, preserving agents, sweetening agents or
flavoring agents. The compositions of the invention may be formulated so as to
provide quick, sustained or delayed release of the active ingredient after
administration to the patient. The compositions are preferably formulated in a unit
dosage form, each dosage containing from about 0.05 mg to about 3 g, more
usually about 64 mg of the active ingredient. However, it will be understood that
the therapeutic dosage administered will be determined by the physician in the
light of the relevant circumstances including the severity of the condition to be
treated, the choice of compound to be administered and the chosen route of
administration. Therefore, the above dosage ranges are not intended to limit the
scope of the invention in any way. The term "unit dosage form" refers to
physically discrete units suitable as unitary dosages for human subjects and other
mammals, each unit containing a predetermined quantity of active material
calculated to produce the desired therapeutic effect, in association with a suitable
pharmaceutical carrier.

In addition to the above formulations, most of which may be administered
orally, the compounds used in the method of the present invention also may be
administered topically. Topical formulations include ointments, creams and gels.

Ointments generally are prepared using either (1) an oleaginous base, i.e.,
one consisting of fixed oils or hydrocarbons, such as white petrolatum or mineral

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oil, or (2) an absorbent base, i.e., one consisting of an anhydrous substance or
substances which can absorb water, for example anhydrous lanolin. Customarily,
following formation of the base, whether oleaginous or absorbent, the active
ingredient (compound) is added to an amount affording the desired concentration.

Creams are oil/water emulsions. They consist of an oil phase (internal
phase), comprising typically fixed oils, hydrocarbons, and the like, such as waxes,
petrolatum, mineral oil, and the like, and an aqueous phase (continuous phase),
comprising water and any water-soluble substances, such as added salts. The two
phases are stabilized by use of an emulsifying agent, for example, a surface active
agent, such as sodium lauryl sulfate; hydrophilic colloids, such as acacia colloidal
clays, veegum, and the like. Upon formation of the emulsion, the active ingredient
(compound) customarily is added in an amount to achieve the desired
concentration.

Gels comprise a base selected from an oleaginous base, water, or an
emulsion-suspension base. To the base is added a gelling agent which forms a
matrix in the base, increasing its viscosity. Examples of gelling agents are
hydroxypropyl cellulose, acrylic acid polymers, and the like. Customarily, the
active ingredient (compounds) is added to the formulation at the desired
concentration at a point preceding addition of the gelling agent.

The amount of compound incorporated into a topical formulation is not
critical; the concentration should be within a range sufficient to permit ready

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application of the formulation to the affected tissue area in an amount which will
deliver the desired amount of compound to the desired treatment site.

The customary amount of a topical formulation to be applied to an affected
tissue will depend upon an affected tissue size and concentration of compound in
the formulation. Generally, the formulation will be applied to the effected tissue in
an amount affording from about 1 to about 500 ^ compound per cm 2 of an
affected tissue. Preferably, the applied amount of compound will range from about
30 to about 300 //g/cm 2 , more preferably, from about 50 to about 200 Atg/cm 2 , and,
most preferably, from about 60 to about 100 Mg/cm 2 .

The following formulation examples are illustrative only and are not
intended to limit the scope of the invention in any way.

Formulation 1

Hard gelatin capsules are prepared using the following ingredients:

Quantity
(mg/capsule)

Active agent 250
starch, dried 200

magnesium stearate IP. _

Total 460 ™g

The above ingredients are mixed and filled into hard gelatin capsules in 460

mg quantities.

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Formulation 2
A tablet is prepared using the ingredients below:

Quantity
(mg/capsule)

Active agent 250
cellulose, microcrystalline 400
5 silicon dioxide, fumed 10

stearic acid 5

Total 665 mg

The components are blended and compressed to form tablets each weighing 665
mg.

Formulation 3

Tablets each containing 60 mg of active ingredient are made as follows:

Quantity
(mg/tablet)

Active agent 60 mg

starch 45 mg

15 microcrystalline cellulose 35 mg

polyvinylpyrrolidone

(as 10% solution in water) 4 mg

sodium carboxymethyl starch 4.5 mg

magnesium stearate 0.5 mg

20 talc 1 mg

Total 150 mg

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The active ingredient, starch and cellulose are passed through a No. 45
mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is
mixed with the resultant powders which are then passed through a No. 14 mesh
U.S. sieve. The granules so produced are dried at 50°C and passed through a No.
1 8 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate and
talc, previously passed through a No. 60 mesh U.S. sieve, are then added to the
granules which, after mixing, are compressed on a tablet machine to yield tablets
each weighing 1 50 mg.
Examples

Kxample 1 . Effects of B rvostatin to PKC isoforms

This experiment demonstrates the dosage and time effects of bryostatin to
PKC isoforms.

Human leukemia cells U937 in the amount of 0.5 x 10 6 were treated with
various amount of bryostatin 1 for 24 hours. Subsequently, the cells were
solubilized for preparation of protein samples according to a routine procedure.
The protein samples from bryostatin treated cells were then used in Western blot
analysis with a protein kinase C-P specific antiserum previously described in Ways
et al., Cell Growth & Differentiation 1994, 5: 1 195-1203. As shown in Figures 1
and 2, bryostatin treatment caused PKC-p activity to decrease within certain
amount of time, i.e., 10 nM bryostatin affects PKC-P within 2 hours, or 1 nM

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bryostatin affects PKC-p within 24 hours. In a repeated experiment, similar results
were obtained.

Example 2. The enhanced efficacy of y-irradiation caused bv PKC-p down
regulation

5 This experiment demonstrates that PKC-P down regulation enhances the

efficacy of y-irradiation.

Human leukemia cells U937 were treated for 24 hours with either 3 nM
bryostatin 1 or the control solution, i.e., the vehicle for bryostatin 1. The cells were
then irradiated with either 500 or 1000 rads of y -irradiation. Seventy-two hours
10 after irradiation, cellular viability was examined using propidium iodide exclusion

and quantified by FACS analysis as previously described in Ways et al., Cell
Growth & Differentiation 1994, 5: 1 195-1203. Viability assays were performed in
triplicate. As shown in Figure 3, y-iiradiation-induced apoptosis was enhanced
under the condition when PKC-P was significantly down-regulated using
15 bryostatin 1 . Similar results were obtained in several repeated experiments.

Example 4. Cells Overexpressing PKC-p Demonstrate Resistance to Radiation
Stimulated Cell Death

Parental U937 cells and U937 PKC-C overexpressing cells (PKC-C cells)
were treated with 0, 500, or 1000 rads of y-irradiation. It is known that PKC-C
20 cells display increased level of PKC-P (Ways et al., Cell Growth & Differentiation,

1 994, 5:1195-1 203). Seventy two hours after irradiation, cellular viability was
examined using propidium iodide exclusion and quantified by FACS analysis as

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previously described in Ways et al., Cell Growth & Differentiation, 1995, 6: 371-
382. Viability assays were performed in triplicate. As shown in Figure 4, cells
having an increased level of PKC-P demonstrated resistance to radiation stimulated
cell death. Similar results were obtained in several repeated experiments.

The principles, preferred embodiments and modes of operation of the
present invention have been described in the foregoing specification. The
invention which is intended to be protected herein, however, is not to be construed
as limited to the particular forms disclosed, since they are to be regarded as
illustrative rather than restrictive. Variations and changes may be made by those
skilled in the art without departing from the spirit of the invention.

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CLAIMS :

1 . A method for treating a neoplasm which comprises administering to
a mammal in need of such treatment, an oncolytic agent having an anti-neoplastic
effect in combination with a protein kinase C inhibitor, wherein the protein kinase
C inhibitor enhances the anti-neoplastic effect of the oncolytic agent.

2. The method of claim 1 wherein the protein kinase C inhibitor is an
inhibitor of the P isozyme of protein kinase C and is a bis-indolylmaleimide or a
macrocyclic bis-indolylmaleimide.

3 . The method of claim 1 wherein the protein kinase C inhibitor is
isozyme selective and where the isozyme selectivity is selected from the group
consisting of beta- 1 and beta-2 isozymes.

4. The method of claim 3 wherein the protein kinase C inhibitor has
the following formula:

R 2

wherein:

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X and Y are independently C,-C 4 alkylene, substituted alkylene, or together
X, Y, and W combine to form -(CH 2 )„-AA-;

R's are hydrogen or up to four optional substituents independently selected
from halo, C,-C 4 alkyl, hydroxy, C ,-C 4 alkoxy, haloalkyl, nitro, NR*R S , or -
NHCO(C,-C 4 alkyl);

R 2 is hydrogen, CH 3 CO-, NH 2 , or hydroxy;

R 3 is hydrogen, (CH 2 ) m aryl, C,-C 4 alkyl, -COO(C r C 4 alkyl), -CONR 4 R 5 , -
(C=NH)NH 2 , -SO(C,-C 4 alkyl), -SO, (NR 4 R 5 ), or -S0 2 (C,-C 4 alkyl);

R 4 and R 5 are independently hydrogen, C,-C 4 alkyl, phenyl, benzyl, or
combine to the nitrogen to which they are bonded to form a saturated or
unsaturated 5 or 6 member ring;

AA is an amino acid residue;

m is independently 0, 1, 2, or 3; and

n is independently 2, 3, 4, or 5, or a pharmaceutically acceptable salt,
prodrug or ester thereof.

5. The method of claim 4 wherein the protein kinase C inhibitor has
the following formula:

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wherein Z is -(CH 2 ) P - or -(CH 2 ) p -0-(CH 2 ) p -; R 4 is hydroxy, -SH, C , -C 4 alkyl,
(CH 2 ) m aryl, -NH(aryl), -N(CH 3 ) (CF 3 ), -NH(CF 3 ), or -NR S R 6 ; R 5 is hydrogen or C
C 4 alky; R 6 is hydrogen, C ,-C 4 alkyl or benzyl; p is 0, 1, or 2; and m is
independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester
thereof.

6. The method of claim 4 wherein the protein kinase C inhibitor has
the following formula:

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wherein Z is -(CH 2 ) P -; R 4 is -NR 3 R 6 , -NH(CF 3 ), or -N(CH 3 ) (CF 3 ); R s and R 6 are
independently H or C,-C 4 alkyl; p is 0, 1 , or 2; and m is independently 2 or 3, or a
pharmaceutically acceptable salt, prodrug or ester thereof.

7. The method of claim 4, wherein the protein kinase C inhibitor
comprises (S)-3,4-[N, N'-l ,l , -((2"-ethoxy)-3'"(0)-4 m -(N,N-dimethylamino)-
butane)-bis-(3,3'-indolyl)]-l(H)-pyrrole-2,5-dione or its pharmaceutically

acceptable acid salt.

8. The method of claim 1 , wherein the oncolytic agent is selected from
the group consisting of Ara-c, VP- 16, cw-platinum, adriamycin, 2-chloro-2-
deoxyadenosine, 9-P-D-arabinosyl-2-fluoroadenine, and glucocorticoids.

9 . A method for treating a neoplasm which comprises administering to
a mammal in need of such treatment, Y-irradiation having an anti-neoplastic effect
in combination with a protein kinase C inhibitor, wherein the protein kinase C
inhibitor enhances the anti-neoplastic effect of Y-irradiation.

10. The method of claim 9 wherein the protein kinase C inhibitor is an
inhibitor of the p isozyme of protein kinase C and is a bis-indolylmaleimide or a
macrocyclic bis-indolylmaleimide.

1 1 . The method of claim 9 wherein the protein kinase C inhibitor is
isozyme selective and where the isozyme selectivity is selected from the group
consisting of beta- 1 and beta-2 isozymes.

12. The method of claim 1 1 wherein the protein kinase C inhibitor has
the following formula:

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R 2

(I)

W is -O-, -S-, -SO-, -S0 2 -, -CO-, C 2 -C 6 alkylene, substituted alkylene, C 2 -
C 6 alkenylene, -aryl-, -aryl(CH 2 ) m O-, -heterocycle-, -heterocycle-(CH 2 ) m O-, -fused
bicyclic-, -fused bicyclic-(CH2) m O-, -NR 3 -, -NOR 3 -, -CONH-, or -NHCO-;

X and Y are independently C,-C 4 alkylene, substituted alkylene, or together
X, Y, and W combine to form -(CH 2 ) n -AA-;

R ! s are hydrogen or up to four optional substituents independently selected
from halo, C,-C 4 alkyl, hydroxy, C ,-C 4 alkoxy, haloalkyl, nitro, NR 4 R 5 , or -
NHCO(C,-C 4 alkyl);

R 2 is hydrogen, CH 3 CO-, NH 2 , or hydroxy;

R 3 is hydrogen, (CH 2 ) m aryl, C r C 4 alkyl, -COO(C r C 4 alkyl), -CONR 4 R 5 , -
(C=NH)NH 2 , -SO(C,-C 4 alkyl), -SO, (NR 4 R S ), or -S0 2 (C,-C 4 alkyl);

R 4 and R s are independently hydrogen, C,-C 4 alkyl, phenyl, benzyl, or
combine to the nitrogen to which they are bonded to form a saturated or
unsaturated 5 or 6 member ring;

AA is an amino acid residue;

m is independently 0, 1, 2, or 3; and

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n is independently 2, 3, 4, or 5, or a pharmaceutically acceptable salt,
prodrug or ester thereof.

1 3 . The method of claim 12 wherein the protein kinase C inhibitor has
the following formula:

wherein Z is -(CH 2 ) P - or -(CH 2 ) p -0-(CH 2 ) p - ; R 4 is hydroxy, -SH, C , -C « alkyl,
(CH 2 ) m aryl, -NH(aryl), -N(CH 3 ) (CF 3 ), -NH(CF 3 ), or -NR*R 6 ; R 5 is hydrogen or C
C 4 alky; R 6 is hydrogen, C ,-C 4 alkyl or benzyl; p is 0, 1, or 2; and m is
independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester
thereof.

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14. The method of claim 12 wherein the protein kinase C inhibitor has
the following formula:

wherein Z is -(CH 2 ) p -; R 4 is -NR S R 6 , -NH(CF 3 ), or -N(CH 3 ) (CF 3 ); R 5 and R 6 are
independently H or C,-C 4 alkyl; p is 0, 1, or 2; and m is independently 2 or 3, or a
pharmaceutically acceptable salt, prodrug or ester thereof.

1 5 . The method of claim 12, wherein the protein kinase C inhibitor
comprises (S)-3,4-[N, N'-l ,1 , -((2 ,, -emoxy)-3"XO)-4 , "-(N,N-dimemylamino)-
butane)-bis-(3,3'-indolyl)]-l(H)-pyrrole-2,5-dione or its pharmaceutically
acceptable acid salt.

WO 97/40842

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1/4

SUBSTITUTE SHEET (RULE 26)

WO 97/40842

PCT/US97/07801

SUBSTITUTE SHEET (RULE 26)

WO 97/40842

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3/4

m L D |C

^ CQ CO

o o

x: ^ o o

<D Z O O

> co in t-

J I I I I I 1 L

o
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CM T3
CO

CO — '
O
CL

SUBSTITUTE SHEET (RULE 26)

WO 97/40842

PCT/US97/07801

4/4

(pe^ejjuou jo %)
AllliqBiA

SUBSTITUTE SHEET (RULE 26)

INTERNATIONAL SEARCH REPORT

International application No.
PCT/US97/07801

A. CLASSIFICATION OF SUBJECT MATTER

IPC(6) :A61K 31/79. 31/40, 33/24
US CL :5 14/34, 411; 424/649
According to International Patent Classification (IPC) or to both national clarification and IPC

B. FIELDS SEARCHED

Minimum documentation searched (claaaification tyitem followed by classification symbols)

U.S. : 514/34,411;424/649

D ocu mentation searched other than minimum documentation to the extent that such documents are included in the fields searched
NONE

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
NONE

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

X, P

US 5,552,396 A (HEATH, JR. ET AL.) 03 September 1996,
see entire document.

1-15

"~j Further documents arc listed in the continuation of Box C. [ | Sec patent family annex.

*L"

■o-

"P-

b*or docuaicat pnhliahrd >Acr the jnjrrwItntTl filing dale off priority

principle or theory uadertynf (be i

o o— ate r ad nwl or cannot be oo— iacrpd to involve bb iaveabve Hap

ooaeidend to Bivocve ea
oombioed with one or ■ore other
beans obvious to a peraoa akilkd

doom

aberof the ■

Date of the actual completion of the international search
22 JULY 1997

Date of mailing of the international search report

2 I AUG 1997

Name and mailing address of the ISA/US
Commiuioaer of Patents and Trademark!
Box PCT

Washington, D.C. 20231
Facsimile No. (703) 305-3230

Authorized officer p. s — ^ /

JEROME D. GOLDBERG yf 1 (^/XJ%^^-y~^~-
TelephoneNo. (703)308-1235

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Full text of "USPTO Patents Application 09993647"

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